In the U.S. in 2015, the poultry industry produced more than 8.5 billion broilers, nearly 100 billion eggs, and over 200 million turkeys. Poultry production is dispersed among all 50 states and the combined value in 2014 was $44.4 billion. There were 299 hatcheries in the US with capacity of over 900 million eggs per month. During 2015, more than 11 billion broiler eggs were set for incubation in hatcheries to yield about 9.2 billion hatched chicks, a loss of about 15%. Of the 9.3 billion hatched chicks, about 9.1 billion are placed into feeding operations (e.g., 97%). Thus, roughly 20% of the two billion eggs placed into hatcheries either fail to hatch or produce chicks that are of insufficient quality for being placed into feeding operations.
Poultry, primarily chickens but also including significant numbers of turkeys, quail, duck, and pheasant, are raised for a variety of reasons. Most poultry is raised for meat and eggs and is supported by a breeding and egg production backend for the continual replacement of layers and broilers. In addition to food production, poultry is also raised and kept as pets. In addition to providing eggs for hatching broilers and layers, eggs are used for table consumption and, importantly, vaccine production. In each situation, there is a need to reduce pathogens of all sorts.
As shown in FIG. 1, the process can be considered to begin with a primary breeding facility that produces several breeds of chickens (breeder chicks) that are provided to breeder farms. The breeder farms produce fertilized eggs that are provided to hatcheries to produce chicks that are in turn destined to be raised as layers (for egg production) and broilers. These breeder farms also provide eggs for hatching and raising by small private farmers and individuals to raise their own chickens and eggs as well as serve as pets. Egg farms maintain laying hens (as well as some roosters) and primarily produce eggs for table consumption and eggs for hatcheries for production of meat (e.g., broilers). Specialized egg farms produce eggs for vaccine production.
Modern poultry production methods generally are vertically integrated systems that include a feed mill which combines corn, soybean meal, and other ingredients, and provides the meal to the breeder farms, grow-out farms, and laying farms. At each stage of the breeding process, whether for broilers (meat) or for egg laying, the population is expanded from 100 to 150 fold. Often, the facilities and processes downstream of the primary breeders are integrated within a single producer and geographical area. Grow-out farms are often contracted by the integrated producer.
Primary breeder companies maintain flocks of inbred lines and provide breeder chicks to breeder farms (from great-grandparent eggs). A single male and 10 females can produce about 150 great-grandparent stock birds (GGP). The GGP birds are bred to produce about 7,500 grandparent (GP) birds that in turn produce 350,000 or so parent stock birds. Parent stock birds in turn produce about 48,000,000 eggs for broiler production (about 120 fold expansion). Breeder farms produce hatching eggs that are transported to the hatchery. After hatching, chicks are graded and sorted and sent to grow-out farms for the production of broilers. Finally, the broilers are sent to processing plants for slaughter, processing, and packaging.
For egg production, the process is similar though modern laying chickens are bred specifically for egg laying. Specialized breeders maintain pure breeding lines and produce hybrid crosses for grandparent stock production which expands the population 100 fold. The grandparent stock (hybrids) is crossed to produce the parent stock, expanding the population 100 fold again (10,000). The parent stock, which can comprise the contributions of four different lines, is used to produce the laying hens, expanding the population another 100 to 120 fold (1,200,000 laying hens). The million or more laying hens produce 600,000,000 eggs that are collected, cooled, cleaned, candled, graded, and packed.
Current production methods begin with collecting and cleaning fertile eggs from breeding farms. Commercial hens begin laying eggs at about 16 to 20 weeks of age, declining rapidly after 25 weeks. While there are various organic and free-range methods, the vast majority of eggs for consumption are obtained from battery cages. More recently, furnished cages (also known as enriched or modified cages) have been developed to improve the welfare of the egg laying hens.
For broilers, production includes maintenance of pedigree stocks for breeding that are pure lines. The eggs are hatched in special pedigree hatcheries and the progeny are used to generate great-grandparent (GGP) and grandparent generations (GP). The GP generation is then supplied to special GP hatcheries to parent stock (PS). The pure, inbred PS lines are then bred in broiler breeder farms from separate male and female lines to produce hybrid offspring. The hybrid offspring are then used for the production of broilers.
Hatcheries receive eggs from egg farms. Certain methods include inoculation of the eggs with antibiotics (typically gentamycin), though an increasing number of producers have production streams that preclude antibiotic use (e.g., products destined to be labeled “no antibiotic”). Increasingly, governments are adopting laws that preclude the routine use of antibiotics in agriculture. Currently, eggs destined for “organic production” can include eggs inoculated with antibiotics.
Whether producing hens for egg laying or broilers for meat production, the fertilized eggs are cleaned and checked for soundness before being placed in incubators. A number of companies manufacture commercial incubators for the large scale production of chicks, including for example, the Jamesway Incubator Company, Inc. (Ontario, Canada), Chickmaster International (Cresskill, N.J.), Natureform Hatcher Technologies (Jacksonville, Fla.) and Surehatch (Cape Town, South Africa). Incubators can be manufactured to incorporate a PHPG generating system or can be modified to retrofit a PHPG generating system as illustrated in Example 12 below. The incubation periods and conditions vary depending on the species and these conditions are well known in the art.
The hatchery typically includes a receiving room to maintain and collect the eggs prior to incubation (on site egg room, receiving room, egg holding-room). The collection and storage at reduced temperatures arrests embryonic development and permits the accumulation of large numbers of eggs for a synchronized hatch. Typically, eggs are stored at 15° C. to 21° C., more usually between 18° C. and 20° C., for between about 24 and 72 hours. See Bourassa et al., “Elevated Egg Holding-Room Temperature of 74° F. (23° C.) does not Depress Hatchability or Chick Quality,” Poultry Science Association, Inc. (2003). Importantly, eggs are stored below the temperature that will arrest further embryonic development (known as “physiological zero” and commonly accepted for chickens as between about 18° C. and 20° C.). Eggs are preferably collected every 30 minutes and transferred to the on-site egg room. During collection and on site storage, eggs are added to the room periodically, typically every couple hours. Eggs are moved from receiving rooms to a first incubator, called a setter, that incubates the eggs at about 37° C., and provides for movement of the eggs to ensure proper development.
The incubation period for chickens is 21 days. For chickens, eggs are incubated in a setter for about 18 days. Following incubation in the setter incubator, the eggs are moved to a hatching incubator where the final incubation period is completed, about 3 to 4 days. Though the incubation and hatching process can be performed in a single incubator, often the incubation and hatching are separated to minimize contamination and decrease costs. Typically, eggs are first incubated in a “setter” for 18 days. After 18 days, the incubated eggs are moved to a hatching incubator where the final three days of incubation are completed or until the hatching process is complete. The chicks are then ready for sorting and shipping to a grow-out farm for broilers or to an egg laying farm as appropriate. As would be understood to one of skill in the art, the increased temperatures and moisture provide a favorable environment for the growth of bacteria. Accordingly, there is a need for methods to reduce the levels of bacteria in both the setter incubator and the hatching incubator (or the combined incubator).
Hatchability is determined by a complex set of factors including storage time, age of the breeders (e.g., laying hens), as well as the incubation conditions. Among the important incubation conditions are the humidity (between 40% and 60% is preferred), temperature (optimal between 37 and 38° C.), turning conditions (eggs need to be turned during setting incubation period), and the gaseous environment (oxygen and carbon dioxide levels). Typically, an optimal breeder farm and hatchery produces about 85 healthy chicks from each 100 eggs set. Yields can commonly be 81% to 82% and can be as low as 70% to 75%. Given the large numbers involved (11 billion eggs set per year), even a small change in hatchability is significant.
After hatching, the chicks are sorted into first-quality chicks (“Q1 chicks”) and second-quality chicks' (“Q2 chicks”). First-quality chicks are boxed and shipped to grow-out farms (broiler farms) where they are raised for meat. Significant losses occur through the hatching stage including dead chicks, second quality chicks, pipped eggs, infertile eggs. Methods of distinguishing Q1 and Q2 chicks are known in the art. See van de Ven et al., “Significance of chick quality score in broiler production,” Animal 6(10):1677-1683 (2012); Tona et al., “Effects of egg storage time on spread of hatch, chick quality and chick juvenile growth,” Poult. Sci. 82(5):736-41 (2003); and Decuypere and Bruggeman, “The endocrine interface of environmental and egg factors affecting chick quality,” Poult. Sci 86(5):1037-42 (2007).
Broiler farms receive chicks from the hatcheries and raise the chicks for meat in “grow-out houses” that are carefully controlled for temperature and humidity. There are about 70,000 grow-out houses located at about 17,000 farms. A typical grow-out house is about 12 to 15 meters wide by 120 to 180 meters long with 2.5 meter high walls. The average area of a grow-out house in the U.S. is about 1600 meters2 (m2), though more recent structures are about 1850 m2. An average grow-out house produces approximately 113,000 birds and 600,000 pounds of meat per year. Like the setting and hatching incubators, the conditions of the grow-out house are critical to the health of the growing birds. Using modern methods, a 2.25 kg bird is produced in about 6 to 7 weeks. Once the growing cycle is complete, the birds are collected for shipment to a processing plant. Processing plants slaughter the broilers, bleed, de-feather and clean the carcass for shipping. Processing can further include chilling and cutting the meat to produce chicken parts.
Further losses occur during the feeding operations which take about 6 weeks from placing to market. Mortality peaks within 3 to 4 days of placement and then declines to a relatively steady level at days 9 or 10. At about day 30, mortality rates begin to rise and peak from between days 40 and 45 until harvest. It has been observed that flocks having high mortality levels in the first week tend to have higher mortality levels at weeks 7 and 8. Thus, methods and conditions that lead to reduced initial mortality are likely to reduce late mortality as well. See Tabler et al., “Mortality Patterns Associated with Commercial Broiler Production,” Avian Advice, 6(1):1-3 (2004).
The causes of poultry mortality and morbidity are varied. Modern commercial poultry production methods often include intensive breeding, hatching, shipping, housing, and processing steps that favor the spread of bacterial, mycobacterial, fungal, parasitic, and viral diseases. In addition to mortality and morbidity, insects also contribute to decreased production.
Modern poultry production methods increase the susceptibility to diseases, including bacterial, viral, and parasitic diseases, due to the intensive nature of the methods. Chickens and other poultry are raised in close proximity to each other and have ample opportunity for direct transmission of diseases. The immune system of birds is different from mammals and may be a contributing factor. For example, it is known that the chicken immune system does not fully develop until well after hatching. In contrast to mammals, birds have hollow bones and thus do not have marrow for producing immune cells. Rather, immune cells are produced in a specialized organ, the bursa. In chickens, the bursa does not fully develop until the chick is about six weeks old, leaving newly hatched chicks particularly vulnerable to infection.
While free range and alternative methods exist, the vast majority of broilers are floor raised indoors on litter such as wood shavings, peanut shells, and rice hulls in buildings called grow-out houses. The housing is climate controlled and provides for feed and water, controlled temperature and moisture, and protects the chicks from predators. Chicks typically reach slaughter weight at about 5 to 9 weeks of age and average about 9 pounds. A typical grow-out house consists of about 20,000 birds.
Given the large number of birds, the presence of the litter, and of course the droppings, the grow-out house faces a number of health challenges. In addition to various insects, parasites, bacteria, and viruses, the droppings and litter produce large amounts of ammonia that are damaging to the chicken's respiratory systems and eyes and can result in hock burns to the legs. Accordingly, grow-out houses are supplied with large amounts of fresh air to remove the ammonia. Improved methods of control in grow-out houses are desirable, including methods that reduce the numbers of insects, parasites, bacteria, and viruses.
The impact of poultry diseases extends beyond the effects on the efficiency and cost of production of poultry products, which are significant. Bacterial diseases by bacteria that are naturally carried by poultry exert an enormous health cost on people. Accordingly, food safety is a priority concern for the poultry industry.
Salmonella, Campylobacter, Listeria, Escherichia coli and Enterococcus are significant causes of disease. The USDA reports that, in 2015, foodborne pathogens resulted in over $10 billion/year in medical costs. As shown in Table 1, Salmonella alone results in $3.7 billion in medical costs. The importance of these bacteria and their link to poultry has generated significant attention to mitigation methods.
TABLE 1Foodborne Illness Medical Costs (2015)DiseaseCostTotal casesHospitalizationsDeathsSalmonella $3.7 billion1,027,56119,336378Toxoplasma gondii $3.3 billion86,6864,428343Listeria monocytogenes $2.8 billion1,5911,173306Norovirus $2.3 billion5,461,73114,663149Campylobacter $1.9 billion845,0248,46376Clostridium perfringens$343 million965,95843826Vibrio vulnificus$320 million969336Yersinia enterocolitica$278 million97,65648029E. coli O157$271 million63,1532,13830Vibrio (non-cholera species)$142 million17,56483See Cost Estimates of Foodborne Illnesses available on the Web at ers.usda.gov/data-products/cost-estimates-of-foodborne-illnesses/
Given the impact of disease on production and human health, the poultry industry and individual operators develop and implement biosecurity plans to prevent the spread of poultry diseases. Biosecurity focuses on preventing the introduction of diseases to a facility and preventing transmission within a facility. Among the priorities are the separation of “clean” and “dirty” areas, provision and use of personal protection equipment (PPE), vector control (insects, worms, rodents, wild birds, pets), equipment control, mortality management, materials management (manure, litter), and feed and material intake control.
Current methods for controlling bacteria attack the problem at different stages of the production process. At the breeder farm or at the hatcher, various methods have been developed to reduce shell surface contamination. At each stage of the production process (Primary Breeder, Breeder Farm, Hatchery, Egg Production), there is an “egg preparation” step that typically involves collecting the eggs soon after laying, washing (dry or wet), fumigation or disinfection, and antibiotic injection to reduce contamination. At the hatchery, combatting bacterial infections has historically relied on antibiotics, though this strategy has increasingly come under scrutiny as it has been theorized the extensive use of antibiotics in agricultural production has contributed to the spread of antibiotic resistance. There exists a need to reduce and eliminate antibiotic use, preferably using organic, non-toxic methods.
The reduction of shell surface contamination has received considerable attention, particularly with regard to bacterial contamination which contributes to human infection and mortality. As noted above, Salmonella as a foodborne illness is costing $3.7 billion/year. Chickens are well known carriers and sources of Salmonella infection and the disease is endemic. Indeed, no method has been identified to eliminate Salmonella from chicken production, in part because Salmonella naturally contaminates healthy chickens. See Humphrey et al., “Numbers of Salmonella enteritidis in the contents of naturally contaminated hens' eggs,” Epidemiol. Infect. 106(3):489-496 (1991). In addition, Salmonella is known to be capable of penetrating egg shells and becoming inaccessible to control methods. See Cox et al., “Salmonella penetration of egg shells and proliferation in broiler hatching eggs—a review,” Poult. Sci 79(11):1571-4 (2000). Cox et al. report that entry of salmonellae can occur through vertical transmission (transmission from an infected hen) and by horizontal transmission after the egg is laid. Current methods rely on injection of antibiotics (usually gentamycin) to reduce bacteria in eggs, but this approach is increasingly disfavored due to linkage to growing antibiotic resistance in bacteria.
Among the methods in use for the reduction of shell surface contamination include fumigation, spray sanitizing methods, UV light irradiation, and egg washing. Fumigation with formaldehyde has been used but has been on the decline due to its potential human toxicity. Several commercial products are available for hatching egg sanitation. See Spray Sanitizing Hatching Eggs from the North Carolina Cooperative Extension Service available on the Web at www.ces.ncsu.edu/depts/poulsci/tech_manuals/spray_sanitizing.html; and Ernst, “Hatching Egg Sanitation: The Key Step in Successful Storage and Production,” ANR Publication 8120 (2004) available on the Web at anrcatalog.ucanr.edu/pdf/8120.pdf.
Among the methods for sanitizing eggs as well as equipment, hydrogen peroxide (H2O2) has shown some promise when eggs are dipped or sprayed with solutions of between 1% and 5%. As a well-known sterilizing agent, hydrogen peroxide has been used to address a number of significant problems in poultry production. Typically, the application of hydrogen peroxide is through the direct application of a solution of hydrogen peroxide (with or without stabilizing agents) by either spraying or dipping. Hydrogen peroxide solutions of 1% or greater are used either alone or in combination with other treatments (e.g., heat or ultraviolet light).
Sheldon and Brake reports that 5% (vol/vol) of H2O2 is required to disinfect shell surfaces. They further report that a first treatment with 2% and a second treatment with 5% H2O2 can improve hatchability. See Sheldon and Brake, “Hydrogen peroxide as an alternative hatching egg disinfectant,” Poult. Sci. 70(5):1092-8 (1991). Padron reported that dipping eggs in 6% H2O2 solution twice reduced the number of Salmonella in eggshell membranes by 95% and reduced the numbers of positive eggs by 55% with no adverse effect on hatchability. See Padron, “Egg dipping in hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes,” Avian Dis. 39(3):627-30 (1995). Bailey et al., report that treatment of broiler eggs with hydrogen peroxide (2.5% fogged) did not significantly reduce, or improve, hatchability. Bailey et al., “Effect of Hatching Cabinet Sanitation Treatments on Salmonella Cross-Contamination and Hatchability of Broiler Eggs,” Poult Sci. 75(2):191-6 (1996). Sander et al., “Effect of hydrogen peroxide disinfection during incubation of chicken eggs on microbial levels and productivity,” Avian Dis. 43(2):227-33 (1999). Cox et al. reported that “hatchability and livability were unaffected by the most effective of the tested treatments.” Cox et al. “Bactericidal Treatment of Hatching Eggs IV. Hydrogen Peroxide Applied with Vacuum and a Surfactant to Eliminate Salmonella from Hatching Eggs,” J. Appl. Poult. Res. 9:530-534 (2000). Cox et al. further report that multiple immersions of hatching eggs further decreased Salmonella with no adverse effect on hatchability. Notably, no improvement to hatchability was reported. Cox et al. studied the use of H2O2 (1.4% solution) applied with a vacuum and a surfactant to reduce Salmonella and reported that after treatment, 30% of the treated eggs remained contaminated. Cox et al., “Bactericidal Treatment of Hatching Eggs V: Efficiency of Repetitive Immersions in Hydrogen Peroxide or Phenol to Eliminate Salmonella from Hatching Eggs,” J. Appl. Poult. Res. 11(3):328-331 (2002). Wells et al. report the combined disinfection of eggshells using ultraviolet light and hydrogen peroxide (3% solution) to achieve up to a 3 log reduction in bacterial counts (cfu/egg). No effects on hatchability or mortality were provided. See Wells et al., “Disinfection of eggshells using ultraviolet light and hydrogen peroxide independently and in combination,” Poult Sci. 89(11):2499-505 (2010). While decreasing bacterial levels, at least temporarily, has not had an adverse effect on hatchability and mortality, neither has H2O2 treatment led to an improvement. None of the studies suggest that lower levels of H2O2 would be effective.
The application and use of H2O2 solutions to poultry production is not without problems however. First, vapor peroxide is highly corrosive and damages the incubator and other equipment in the area, even after a single use. The hatcheries don't like using it for that reason. Second, the aqueous and vapor peroxide treatments simply shock the bacteria on the eggs, reducing the log count, but not fully eliminating the bacteria; thus, once the eggs are placed in incubation (e.g., optimal growth conditions) the bacteria can quickly regrow to pre-treatment levels. Third, vapor or liquid hydrogen peroxide may harm the integrity of the eggshells, which are calcium carbonate, making them more permeable to bacteria that regrow on the eggs after treatment. Thus, the regrown bacteria may potentially have more impact through compromised eggshells. Fourth, a single droplet of vaporized or nebulized hydrogen peroxide can have 108 to 109 molecules of H2O2 per cubic micron. Such levels are toxic and cannot be used in occupied areas. For at least these reasons, eggs destined for hatching are not treated with hydrogen peroxide solutions.
There are a number of alternatives to hydrogen peroxide available. Mueller-Doblies et al., report that “disinfectants containing a mixture of formaldehyde, glutaraldehyde and QAC perform significantly better under field conditions than oxidizing products and should therefore be the first choice for disinfection of turkey premises where Salmonella is present.” Mueller-Doblies et al. “A comparison of the efficacy of different disinfection methods in eliminating Salmonella contamination from turkey houses,” J. Appl. Microbiol. 109(2):471-479 (2010) (“[D]isinfectants containing a mixture of formaldehyde, glutaraldehyde and QAC perform significantly better under field conditions than oxidizing products and should therefore be the first choice for disinfection of turkey premises where Salmonella is present”). Another approach is the application of chlorine dioxide either alone or with heat. This approach, like the use of aerosolized H2O2, formaldehyde, and glutaraldehyde is toxic and cannot be used in occupied areas. See, Kim et al., “Inactivation of Salmonella on Eggshells by Chlorine Dioxide Gas,” Korean J. Food Sci. Anim. Resour. 36(1):100-108 (2016); Park et al., “Inactivation of Salmonella enterica in chicken feces on the surface of eggshells by simultaneous treatments with gaseous chlorine dioxide and mild wet heat,” Food Microbiol. 62:202-206 (2017); and Choi et al., “Reduction of Salmonella enterica on the surface of eggshells by sequential treatment with aqueous chlorine dioxide and drying,” Int. J. Food Microbiol. 210:84-87 (2015).
In addition, it has been shown that H2O2 treatments can have negative effects. Nebulized (e.g., vaporized) hydrogen peroxide is known to increase the susceptibility of chickens to avian pathogenic Escherichia coli (APEC), a cause of colibacillosis in chickens at all ages. Nebulized hydrogen peroxide is a mist comprising droplets of hydrogen peroxide at the indicated concentration and commercial nebulizers can prepare droplets of about 1 to 5 micrometers (μm) in diameter. See European Patent Publication No. EP 2 644 282. As provided by Oosterik et al., the “worsening effect [of increased bacterial lesions] after nebulization is probably due to the caustic effect of H2O2 radicals on (ciliated) epithelial cells . . . .” Oosterik et al., “Disinfection by hydrogen peroxide nebulization increases susceptibility to avian pathogenic Escherichia coli,” BMC Res. Notes. 8:378 (2015). A droplet of vaporized H2O2 contains 100,000,000 to 1,000,000,000 molecules of H2O2 compared to the 5 to 25 molecules of DHP per cubic micron of air. Thus, the literature teaches the cautious application of hydrogen peroxide when applied to living cells as it can have significant negative consequences.
In addition to being a well-known sterilant, hydrogen peroxide is also known to be involved in cellular homeostasis and part of an inductive signaling process. For example, Patterson et al. report that hydrogen peroxide-regulated homeostasis involves the tyrosine-protein kinase lyn and the tyrosine-protein kinase syk. Both lyn and syk are known to be involved in signaling in mouse and chicken cells and in particular hematopoietic and nonhematopoietic cells and are proto-oncogenes. Hydrogen peroxide is also known to induce programmed cell death (apoptosis) in many cell types and organisms. See Wan et al, “Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis,” PLoS One 11(1):e0147950 (2016). Hydrogen peroxide is also known to play an important role in angiogenesis. At low concentrations, H2O2 stimulates proliferation and migration and inhibits at higher concentrations. At higher concentrations, H2O2 induces new vessel formation; while at even higher concentrations, it induces apoptosis. See Mu et al., “Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase,” Cell Biol. Int. 34(10):1013-1020 (2010). In addition to effects on cells, hydrogen peroxide is also known to affect the porosity of eggshell membranes. See Hsieh et al., “Hydrogen peroxide treatment of eggshell membrane to control porosity,” Food Chem. 141(3):2117-2121 (2013). Thus, the application of DHP gas to poultry and more particularly to eggs during development could result in significant, and unpredictable, changes in poultry development and health.
Another important use of eggs, primarily chicken, is for the production of vaccines, typically influenza. Vaccine producers receive eggs from specialized egg farms and incubate the eggs for a short period. Given the sensitive nature of vaccine production, even a single poultry pathogen can cause the ruination of an entire batch of vaccine eggs, causing upwards of a million dollars and six weeks in loss. The delay in vaccine production in turn can result in deaths due to delays in flu vaccination.
For vaccine production in particular, there is a need for reduced levels of contamination. Vaccine production facilities typically receive eggs from smaller farms with lower contamination levels. These eggs are incubated in the same types of incubators used in hatcheries, but on a smaller scale. Each small batch of eggs (tens of thousands) is worth millions of dollars and one contaminated egg can ruin an entire batch. Thus, there is a need to further reduce pathogen contamination of eggs for vaccine production.
In view of these significant losses, even small increases in incubation to placement efficiency can result in significant savings and increased profitability for hatcheries.
Aqueous hydrogen peroxide (H2O2) is a strong oxidant and has well known antimicrobial and antiseptic properties, as well as activity against organic compounds. H2O2 also has activity against volatile organic compounds (VOCs) oxidizing them and hydrolyzing them and breaking them down. Hydrogen peroxide hydrolyzes, among other things, formaldehyde, carbon disulfide, carbohydrates, organophosphorus and nitrogen compounds, and many other more complex organic molecules. H2O2 is produced commercially in large quantities as either a colorless liquid or as an aqueous solution, generally from about 3% to 90%. See Merck Index, 10th Edition at 4705 to 4707. It has recently been shown that H2O2 can be produced as a purified hydrogen peroxide gas (PHPG) that is free of ozone, plasma species, or organic species.
PHPG is a non-hydrated gaseous form of H2O2 that is distinct from liquid forms of hydrogen peroxide, including hydrated aerosols and vaporized forms. PHPG is generated in situ from ambient water vapor and cannot be produced from a solution of hydrogen peroxide. Aerosolized and vaporized forms of hydrogen peroxide solution have significantly higher concentrations of H2O2, typically comprising greater than 1×106 molecules per cubic micron compared to air containing PHPG that contains between 5 and 25 molecules per cubic micron. Hydrogen peroxide aerosols and vapors are prepared from aqueous solutions of hydrogen peroxide and also differ from PHPG as the aerosols are hydrated and, regardless of the size of the droplet, settle under the force of gravity. Vaporized forms condense and settle. Aerosolized forms of hydrogen peroxide are effective antimicrobial agents; however, they are generally considered toxic and wholly unsuitable for use in occupied spaces. See for example, Kahnert et al., “Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis,” Lett. Appl. Microbiol. 40(6):448-52 (2005). The application of vaporized hydrogen peroxide has been limited by concerns of explosive vapors, hazardous reactions, corrosivity, and worker safety. See Agalloco et al., “Overcoming Limitations of Vaporized Hydrogen Peroxide,” Pharmaceutical Technology, 37(9):1-7 (2013). Further, spaces treated with aerosolized forms, typically at concentrations of between 150 and 700 ppm, remain unsuitable for occupation until the H2O2 has been reduced by degradation to water and oxygen and the H2O2. The use of PHPG solves the problem of toxicity of aerosolized H2O2 (e.g., vaporized and liquid forms of H2O2) and can provide continuous safe antimicrobial and oxidative activity.
PHPG is non-hydrated and behaves essentially as an ideal gas. In this form, PHPG behaves largely as an ideal gas and is capable of diffusing freely throughout an environment to attain an average concentration of about 25 molecules per cubic micron of air. As a gas, PHPG is capable of penetrating most porous materials, essentially diffusing freely to occupy any space that is not airtight. The gaseous form of hydrogen peroxide doesn't settle, deposit, or condense when present at concentrations at least up to 10 ppm. PHPG is completely “green” and leaves no residue as it breaks down the water and oxygen. PHPG cannot be prepared from an aqueous solution even if the vaporized form is a so-called “dried” form.
Importantly, and in contrast to vaporized and aerosolized forms of H2O2, environments containing up to 1 ppm H2O2 have been designated as safe for continuous human occupation under current Occupational Safety and Health Administration (OSHA), National Institute for Occupational Safety and Health (NIOSH), or American Conference of Industrial Hygienists (ACGIH) standards. It is believed that 10 ppm is also safe for human occupation, though not yet recognized by the regulatory authorities. It is further anticipated that up to 50 ppm of PHPG is safe, but that level has not been tested. With the advent of PHPG generating devices, appropriate studies can now be performed. The ability to produce effective amounts of PHPG and the safety of PHPG when present as a dry hydrogen peroxide (DHP) gas combined with its effectiveness as an antimicrobial agent, suggests a myriad of potentially useful applications remain to be discovered.
U.S. Pat. No. 8,168,122 issued May 1, 2012, and U.S. Pat. No. 8,685,329 issued Apr. 1, 2014, both to Lee, disclose methods and devices to prepare PHPG for microbial control and/or disinfection/remediation of an environment (e.g., solid surfaces). International Patent Application No. PCT/US2014/038652, published as International Patent Publication No. WO 2014/186805, discloses the effectiveness and use of PHPG for the control of arthropods, including insects and arachnids. International Patent Application No. PCT/US2014/051914, published Feb. 26, 2015, as International Patent Publication No. WO2015/026958, discloses the beneficial effects of PHPG on respiratory health, including increased resistance to infection and increased hypothiocyanate ions in mammalian lungs. International Patent Application No. PCT/US2015/029276, published Nov. 12, 2015, as International Patent Publication No. WO 2015/171633, discloses improved PHPG generating devices. International Patent Application No. PCT/US2016/028457, published Oct. 27, 2016, as International Patent Publication No. WO 2016/172223, discloses an application of DHP to clean rooms. International Patent Application No. PCT/US2016/029847, published Nov. 3, 2016, as International Patent Publication No. WO 2016/176486, discloses methods of use of DHP in agricultural production, transport, and storage. The contents of each of the foregoing applications are incorporated herein by reference in their entireties.
While not limited, the present specification provides for the first time the effects of PHPG/DHP treatment on eggs and poultry. In previous studies, PHPG/DHP was demonstrated to be effective on solid surfaces. In other studies DHP was shown to be beneficial and therapeutic to people and yet toxic to arthropods; thus, it was unclear how DHP would affect egg and chick development.
It is provided here that porous, gas permeable egg shells can be effectively treated with DHP and embryos undergo normal development. It is further provided that eggs can be safely and effectively treated at laying and safely transferred to climate controlled on site egg rooms. The present application provides that the continuous treatment of eggs through the setting and hatching stages is safe and results in improvements to chick health. Notable improvements include, but are not limited to, increased hatchability, decreased post hatching mortality, improved food conversion ratio, reduced cull rates, and increased weight at hatching. Prior to the present disclosure, it was unclear whether DHP would be effective against microbes on the surface and presumably within pores. More importantly, prior to the present disclosure, the safety of DHP on the developing embryos was uncertain. Here we show that DHP is not only safe for extended application to poultry eggs but also significantly improves the quality of the egg including, for example, reducing bacterial loads, reducing the number of rotten eggs, decreasing 1 week mortality, and decreasing overall mortality. In addition, application of DHP during the laying, storing, incubating, and hatching stages decreases deformities and increases hatch rates.
As disclosed, the poultry production process consists of numerous steps that provide for exposure of the poultry to pathogens. Not to be limited by theory or example, DPH treatment provides for reduced contamination at egg laying, reduced bacterial loads and growth during on-site storage, prevention of contamination of compromised eggs (e.g., eggs with microcracks), reduced seven day mortality, decreased cull rates, reduced rates of condemnation, and reduced on-farm mortality. The safety and efficacy of hydrogen peroxide gas allows for each step in the process to be targeted separately and as part of an overall mitigation strategy to reduce viral and bacterial pathogens, to reduce parasitic pathogens, and to reduce the various insect vectors that transmit pathogens.